Project Summary/Abstract Prosthetic grafts are used widely in vascular reconstructive surgery, but their long-term patency is limited by their thrombogenicity and the development of intimal hyperplasia. Oxidized LDL and lysophosphatidylcho- line, a product of LDL oxidation, accumulate in grafts and alter cell function. The long-term goal of our research is to improve the patency of vascular grafts by promoting endothelial cell (EC) healing of graft surfaces. LysoPC inhibits EC migration in vitro, and hypercholesterolemia reduces EC migration into injured arteries and onto grafts. OxLDL and lysoPC increase cellular production of reactive oxygen species, increase cell mem- brane fluidity, and open ion channels. These effects can inhibit EC migration. Specifically, lysoPC activates a canonical transient receptor potential (TRPC) ion channel, TRPC6, which opens TRPC5 through a unique TRPC activation cascade, leading to a prolonged rise in intracellular free calcium ion concentration ([Ca2+]i). Increased [Ca2+]i inhibits EC migration by activation of calpains that breakdown cytoskeletal proteins essential for migration. This proposal addresses the hypothesis that lipid oxidation products formed within synthetic vascular grafts inhibit their EC migration, in part through activation of TRPC5 and TRPC6 channels, and thereby limit endothelialization of vascular grafts in vivo. The goals of this project are to identify mechanisms by which lipid oxidation products activate TRPC 5 and TRPC6 channels and identify ways to counteract this. To accomplish these goals, the mechanism by which lipid oxidation products activate TRPC6, specifically the roles of Src kinases and phospholipase C-g1, will be explored. In addition, and the mechanism by which TRPC6 activates TRPC5 will be studied, focusing on the role of intracellular calcium and myosin light chain kinase. The role of reactive oxygen species and changes in membrane fluidity in these actions will also be explored. Finally, the ability of HDL to improve EC migration in areas of arterial injury in mice and onto pros- thetic grafts implanted in normal and hypercholesterolemic rabbits will be assessed. The proposed studies will investigate a mechanism by which lipid oxidation products limit EC healing of vascular injuries and synthetic vascular grafts. Studies will also address the ability of HDL to promote EC healing. These studies will lead to a better understanding of the role of lipids in the pathophysiology of graft failure, and ultimately, to methods promoting endothelial healing of angioplasty sites and prosthetic grafts to prolong their patency for the benefit of all people undergoing cardiovascular interventions.